Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

Abstract

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

Description

[0002] This invention relates generally to the field of nucleic acid hybridization on membranes. More particularly, this invention relates to a method for automated multiplex sequencing of DNA.[0003] Large scale nucleotide sequencing initiatives, such as a project to sequence the human genome, have created a need for increased efficiency and productivity. J. Watson, 248 Science 44 (1990). Automation of the various steps involved in sequencing is one area in which gains in efficiency and productivity are being made.[0004] Multiplex sequencing, one scheme for reducing the number of sequencing reactions and electrophoresis steps, involves the processing of a mixture of sequencing templates followed by sequential hybridization with selected probes. G. Church & S. Kieffer-Higgins, 240 Science 185 (1988); U.S. Pat. No. 4,942,124. In this method, many sequencing templates, each carrying a short known sequence or tag, are processed together. A single DNA preparation yields a mixture of temp...

AI Technical Summary

Benefits of technology

[0022] Another aspect of the invention is providing a fluorogenic substrate that is converted into a fluorescent product by an enzyme, wherein enzyme turnover produces many copies of the fluorescent product, the fluorescent product produces a clear pattern of hybridization with the support-bound nucleic acid, and the fluorescent product is easily removed for subsequent rounds of hybridization. Illustrative of suitable fluorogenic substrates is the benzothiazole derivative, 2'-(2-benzothiazolyl)-6'-hydroxybenzothiazole phosphate (BBTP)-Alkaline phosphatase catalyzes the conversion of BBTP into the fluorescent product BBT. BBT does not diffuse on nylon membranes thus providing a sharp fluorescent image of membrane-bound DNA when illuminated with a wavelength of light that excites fluorescence. BBT is easily removed from the membrane by washing in detergent so that subsequent hybridizations can be performed on the same membrane.

Problems solved by technology

The savings made in sequencing reactions and electrophoresis by multiplex sequencing are offset to some extent, however, by new steps that are unnecessary in conventional sequencing protocols.

A remaining problem is the acquisition of sequence data in electronic form.

Although radioactive probes can detect minute g quantities of DNA, they are hazardous and unstable, and high-resolution direct imaging of radioactive signals is not straight-forward.

Although calorimetric detection of sequence ladders has been achieved, P. Richterich et al, 7 Bio / Techniques 52 (1989), the inability to remove the colored product from the membrane precludes its use for sequential probing.

Low fluorescence membranes, such as amine derivatized polypropylene (e, g., U.S. Pat. No. 5,112,736), are known, however such low flourescence membranes are restricted by a limit of detection about 100-fold too high for multiplex sequencing and the membranes are more fragile than nylon membranes.

However the light output from chemiluminescence is quite low.

Due to the low level of light emitted, a sensitive, low-noise detector, such as a cryogenically cooled CCD, is required for imaging, and a long exposure time is needed.

A fully automated system based on chemiluminescence could be constructed, but it would be expensive and slow.

When compared to other methods of visualization, however, such as autoradiography using isotope labels and X-ray film, the most obvious limitation of CCD imaging lies in the dimensions of the sensor arrays most commonly used in analytical applications.

Their limited size rules out the recording of high-resolution electropherograms on a single frame.

However, this procedure is time consuming and labor intensive, and the quality of the resulting composite image is compromised by discontinuities.

CCD arrays consisting of 2048 elements square are commercially available, although at prices that are often prohibitive for analytical applications.

The task of converting relative band positions into nucleotide sequence is conceptually simple, however, the 1-3% error rate of human readers indicates that reading is more complex in practice.

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